Polyphosphoinositides (PPI) play crucial roles in cellular signaling and functions. However, comprehensively determining the changed levels of these species during different cellular processes has faced difficulties. Herein, we applied a novel methylation pattern recognition and simulation approach, and we exploited newly derived fragmentation patterns of methylated PPI species for comprehensive analysis of PPI species including phosphate position(s) and fatty acyl chains capable of circumpassing previous limitations. The developed method was applied for quantitative analysis of PPI species present in diabetic mouse cortex and liver, and it allowed us to unravel the marked reduction of PPI levels in brain cortices of db/db mice for the first time. Taken together, we developed a powerful and high-throughput method for comprehensive analysis of PPI species, which should greatly contribute to the elucidation of PPI biology under different disease states.